Traditional Cultivation and Identification

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Traditional Cultivation and Identification

Direct laboratory methods such as microscopy provide preliminary information about the bacteria involved in an infection, but bacterial growth is usually required for definitive identification and characterization. This chapter presents the various principles and methods required for bacterial cultivation and identification.

Principles of Bacterial Cultivation

This section focuses on the principles and practices of bacterial cultivation, which has three main purposes:

Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection site (i.e., the in vivo environment) by some means of specimen collection and growing them in the artificial environment of the laboratory (i.e., the in vitro environment). Once grown in culture, most bacterial populations are easily observed without microscopy and are present in sufficient quantities to allow laboratory identification procedures to be performed.

The successful transition from the in vivo to the in vitro environment requires that the nutritional and environmental growth requirements of bacterial pathogens be met. The environmental transition is not necessarily easy for bacteria. In vivo they are utilizing various complex metabolic and physiologic pathways developed for survival on or within the human host. Then, relatively suddenly, they are exposed to the artificial in vitro environment of the laboratory. The bacteria must adjust to survive and multiply. Of importance, their survival depends on the availability of essential nutrients and appropriate environmental conditions.

Although growth conditions can be met for most known bacterial pathogens, the needs of certain clinically relevant bacteria are not sufficiently understood to allow for development of in vitro laboratory growth conditions. Examples include Treponema pallidum (the causative agent of syphilis) and Mycobacterium leprae (the causative agent of leprosy).

Nutritional Requirements

As discussed in Chapter 2, bacteria have numerous nutritional needs that include different gases, water, various ions, nitrogen, sources for carbon, and energy. The source for carbon and energy is commonly supplied in carbohydrates (e.g., sugars and their derivatives) and proteins.

General Concepts of Culture Media

In the laboratory, nutrients are incorporated into culture media on or in which bacteria are grown. If a culture medium meets a bacterial cell’s growth requirements, then that cell will multiply to sufficient numbers to allow visualization by the unaided eye. Of course, bacterial growth after inoculation also requires that the medium be placed in optimal environmental conditions.

Because different pathogenic bacteria have different nutritional needs, various types of culture media have been developed for use in diagnostic microbiology. For certain bacteria, the needs are relatively complex, and exceptional media components must be used for growth. Bacteria with such requirements are said to be fastidious. Alternatively, the nutritional needs of most clinically important bacteria are relatively basic and straightforward. These bacteria are considered nonfastidious.

Phases of Growth Media

Growth media are used in either of two phases: liquid (broth) or solid (agar). In some instances (e.g., certain blood culture methods), a biphasic medium that contains both a liquid and a solid phase may be used.

In broth media, nutrients are dissolved in water, and bacterial growth is indicated by a change in the broth’s appearance from clear to turbid (i.e., cloudy). The turbidity, or cloudiness, of the broth is due to light deflected by bacteria present in the culture (Figure 7-1). More growth indicates a higher cell density and greater turbidity. At least 106 bacteria per milliliter of broth are needed for turbidity to be detected with the unaided eye.

In addition to amount of growth present, the location of growth within thioglycollate broth indicates the type of organism present based on oxygen requirements. Strict anaerobes will grow at the bottom of the broth tube, whereas aerobes will grow near the surface. Microaerophilic organisms will glow slightly below the surface where oxygen concentrations are lower than atmospheric concentrations. In addition, facultative anaerobes and aerotolerant organisms will grow throughout the medium, as they are unaffected by the variation in oxygen content.

A solid medium is a combination of a solidifying agent added to the nutrients and water. Agarose, the most common solidifying agent, has the unique property of melting at high temperatures (≥95° C) but re-solidifying only after its temperature falls below 50° C. The addition of agar allows a solid medium to be prepared by heating to an extremely high temperature, which is required for sterilization and cooling to 55° C to 60° C for distribution into petri dishes. On further cooling, the agarose-containing medium forms a stable solid gel referred to as agar. The petri dish containing the agar is referred to as the agar plate. Different agar media usually are identified according to the major nutritive components of the medium (e.g., sheep blood agar, bile esculin agar, xylose-lysine-desoxycholate agar).

With appropriate incubation conditions, each bacterial cell inoculated onto the agar medium surface will proliferate to sufficiently large numbers to be observable with the unaided eye (see Figure 7-1). The resulting bacterial population is considered to be derived from a single bacterial cell and is known as a pure colony. In other words, all bacterial cells within a single colony are the same genus and species, having identical genetic and phenotypic characteristics (i.e., are derived from a single clone). Pure cultures are required for subsequent procedures used to identify and characterize bacteria. The ability to select pure (individual) colonies is one of the first and most important steps required for bacterial identification and characterization.

Media Classifications and Functions

Media are categorized according to their function and use. In diagnostic bacteriology there are four general categories of media: enrichment, nutritive, selective, and differential.

Enrichment media contain specific nutrients required for the growth of particular bacterial pathogens that may be present alone or with other bacterial species in a patient specimen. This media type is used to enhance the growth of a particular bacterial pathogen from a mixture of organisms by providing specific nutrients for the organism’s growth. One example of such a medium is buffered charcoal-yeast extract agar, which provides l-cysteine and other nutrients required for the growth of Legionella pneumophila, the causative agent of legionnaires’ disease (Figure 7-2).

Enrichment media may also contain specialized enrichment broths used to enhance the growth of organisms present in low numbers. Broths may be used to ensure growth of an organism when no organisms grow on solid media following initial specimen inoculation. Enrichment broths used in the clinical laboratory often include thioglycollate for the isolation of anaerobes, LIM broth for selective enrichment of group B streptococci, and gram-negative (GN) broth for the selective enrichment of enteric gram-negative organisms.

Nutritive media or supportive media contain nutrients that support growth of most nonfastidious organisms without giving any particular organism a growth advantage. Nutrient media include tryptic soy agar, or nutrient agar plates for bacteria or Sabouraud’s dextrose agar for fungi. Selective media contain one or more agents that are inhibitory to all organisms except those “selected” by the specific growth condition or chemical. In other words, these media select for the growth of certain bacteria to the disadvantage of others. Inhibitory agents used for this purpose include dyes, bile salts, alcohols, acids, and antibiotics. An example of a selective medium is phenylethyl alcohol (PEA) agar, which inhibits the growth of aerobic and facultatively anaerobic gram-negative rods and allows gram-positive cocci to grow (Figure 7-3). Selective and inhibitory chemicals included within nutritive media prevent the overgrowth of normal flora or contaminating organisms that would prevent the identification of pathogenic organisms. However, it is important to note that the use of selective media does not ensure that the inhibited organisms are not present in small quantity and may simply be too small to see.

Differential media employ some factor (or factors) that allows colonies of one bacterial species or type to exhibit certain metabolic or culture characteristics that can be used to distinguish it from other bacteria growing on the same agar plate. One commonly used differential medium is MacConkey agar, which differentiates between gram-negative bacteria that can and cannot ferment the sugar lactose (Figure 7-4).

Of importance, many media used in diagnostic bacteriology provide more than one function. For example, MacConkey agar is both differential and selective or combination media because it will not allow most gram-positive bacteria to grow. Another example is sheep blood agar. This is the most commonly used nutritive medium for diagnostic bacteriology because it allows many organisms to grow. However, in many ways this agar is also differential because the appearance of colonies produced by certain bacterial species is readily distinguishable, as indicated in Figure 5-2. Figure 7-5 shows differential hemolytic patterns by various organisms.

Summary of Artificial Media for Routine Bacteriology

Various broth and agar media that have enrichment, selective, or differential capabilities and are used frequently for routine bacteriology are listed alphabetically in Table 7-1. Anaerobic bacteriology (Section 13), mycobacteriology (Section 14), and mycology (Chapter 60) use similar media strategies; details regarding these media are provided in the appropriate chapters.


Plating Media for Routine Bacteriology

Medium Components/Comments Primary Purpose
Bile esculin agar (BEA) Nutrient agar base with ferric citrate. Hydrolysis of esculin by group D streptococci imparts a brown color to medium; sodium desoxycholate inhibits many bacteria Differential isolation and presumptive identification of group D streptococci and enterococci
Bile esculin azide agar with vancomycin Contains azide to inhibit gram-negative bacteria, vancomycin to select for resistant gram-positive bacteria, and bile esculin to differentiate enterococci from other vancomycin-resistant bacteria that may grow Selective and differential for cultivation of vancomycin-resistant enterococci from clinical and surveillance specimens
Blood agar Trypticase soy agar, Brucella agar, or beef heart infusion with 5% sheep blood Cultivation of nonfastidious microorganisms, determination of hemolytic reactions
Bordet-Gengou agar Potato-glycerol–based medium enriched with 15%-20% defibrinated blood; contaminants inhibited by methicillin (final concentration of 2.5 µm/mL) Isolation of Bordetella pertussis and Bordetella parapertussis
Brain heart infusion agar or broth Dextrose, pork brain and heart dehydrated infusions. Cultivation of fastidious organisms.
Buffered charcoal-yeast extract agar (BCYE) Yeast extract, agar, charcoal, and salts supplemented with L-cysteine HCl, ferric pyrophosphate, ACES buffer, and α-ketoglutarate Enrichment for Legionella spp.
Supports the growth of Francisella and Nocardia spp.
Buffered charcoal-yeast extract (BCYE) agar with antibiotics BCYE supplemented with polymyxin B, vancomycin, and ansamycin, to inhibit gram-negative bacteria, gram-positive bacteria, and yeast, respectively Enrichment and selection for Legionella spp.
Burkholderia cepacia selective agar Bile salts, gentamycin, ticarcillin, polymixin B, Peptone, yeast extract For recovery of B. Cepacia from cystic fibrosis patients
Campy-blood agar Contains vancomycin (10 mg/L), trimethoprim (5 mg/L), polymyxin B (2500 U/L), amphotericin B (2 mg/L), and cephalothin (15 mg/L) in a Brucella agar base with sheep blood Selective for Campylobacter spp.
Campylobacter thioglycollate broth Thioglycollate broth supplemented with increased agar concentration and antibiotics Selective holding medium for recovery of Campylobacter spp.
Incubated at 4° C for cold-enrichment.
CDC anaerobe 5% sheep blood agar Tryptic soy broth, 5% sheep blood and added nutrients Improved growth of obligate, slow-growing anaerobes
Cefoperazone, vancomycin, amphotericin (CVA) medium Blood-supplemented enrichment medium containing cefoperazone, vancomycin, and amphotericin to inhibit growth of most gram-negative bacteria, gram-positive bacteria, and yeast, respectively Selective medium for isolation of Campylobacter spp.
Cefsulodin-irgasan-novobiocin (CIN) agar Peptone base with yeast extract, mannitol, and bile salts; supplemented with cefsulodin, irgasan, and novobiocin; neutral red and crystal violet indicators Selective for Yersinia spp.; may be useful for isolation of Aeromonas spp.
Chocolate agar Peptone base, enriched with solution of 2% hemoglobin or IsoVitaleX (BBL) Cultivation of fastidious microorganisms such as Haemophilus spp., Brucella spp. and pathogenic Neisseria spp.
Chromogenic media Organism-specific nutrient base, selective supplements and chromogenic substrate Chromogenic media are designed to optimize growth and differentiate a specific type of organism. Chromagars are routinely used in the identification of yeasts, methicillin-resistant Stapylococcus aureus (MRSA), and a variety of other organisms.
Columbia colistin-nalidixic acid (CNA) agar Columbia agar base with 10 mg colistin per liter, 15 mg nalidixic acid per liter, and 5% sheep blood Selective isolation of gram-positive cocci
Cystine-tellurite blood agar Infusion agar base with 5% sheep blood; reduction of potassium tellurite by Corynebacterium diphtheriae produces black colonies Isolation of C. diphtheriae
Eosin methylene blue (EMB) agar (Levine) Peptone base containing lactose; eosin Y and methylene blue as indicators Isolation and differentiation of lactose-fermenting and non–lactose-fermenting enteric bacilli
Gram-negative broth (GN) Peptone base broth with glucose and mannitol; sodium citrate and sodium desoxycholate act as inhibitory agents Selective (enrichment) liquid medium for enteric pathogens
Hektoen enteric (HE) agar Peptone base agar with bile salts, lactose, sucrose, salicin, and ferric ammonium citrate; indicators include bromthymol blue and acid fuchsin Differential, selective medium for the isolation and differentiation of Salmonella and Shigella spp. from other gram-negative enteric bacilli
Loeffler’s medium Animal tissue (heart muscle), dextrose, eggs and beef serum, and sodium chloride Isolation and growth of Corynebacterium
MacConkey agar Peptone base with lactose; gram-positive organisms inhibited by crystal violet and bile salts; neutral red as indicator Isolation and differentiation of lactose fermenting and non–lactose-fermenting enteric bacilli
MacConkey sorbitol agar A modification of MacConkey agar in which lactose has been replaced with d-sorbitol as the primary carbohydrate For the selection and differentiation of E. coli O157:H7 in stool specimens
Mannitol salt agar Peptone base, mannitol, and phenol red indicator; salt concentration of 7.5% inhibits most bacteria Selective differentiation of staphylococci
New York City (NYC) agar Peptone agar base with cornstarch, supplemented with yeast dialysate, 3% hemoglobin, and horse plasma; antibiotic supplement includes vancomycin (2 µg/mL), colistin (5.5 µg/mL), amphotericin B (1.2 µg/mL), and trimethoprim (3 µg/mL) Selective for Neisseria gonorrhoeae;
also supports the growth of Ureaplasma urealyticum and some Mycoplasma spp.
Phenylethyl alcohol (PEA) agar Nutrient agar base. Phenylmethanol inhibits growth of gram-negative organisms Selective isolation of aerobic gram-positive cocci and bacilli and anaerobic gram-positive cocci and negative bacilli
Regan Lowe Charcoal agar supplemented with horse blood, cephalexin, and amphotericin B Enrichment and selective medium for isolation of Bordetella pertussis
Salmonella-Shigella (SS) agar Peptone base with lactose, ferric citrate, and sodium citrate; neutral red as indicator; inhibition of coliforms by brilliant green and bile salts Selective for Salmonella and some Shigella spp.
Schaedler agar Peptone and soy protein base agar with yeast extract, dextrose, and buffers; addition of hemin, l-cystine, and 5% blood enriches for anaerobes Nonselective medium for the recovery of anaerobes and aerobes
Selective for Campylobacter and Helicobacter spp.
Selenite broth Peptone base broth; sodium selenite toxic for most Enterobacteriaceae Enrichment of isolation of Salmonella spp.
Skirrow agar Peptone and soy protein base agar with lysed horse blood; vancomycin inhibits gram-positive organisms; polymyxin B and trimethoprim inhibit most gram-negative organisms Selective for Campylobacter spp.
Streptococcal selective agar (SSA) Contains crystal violet, colistin, and trimethoprim- sulfamethoxazole in 5% sheep blood agar base Selective for Streptococcus pyogenes and Streptococcus agalactiae
Tetrathionate broth Peptone base broth; iodine and potassium iodide, bile salts, and sodium thiosulfate inhibit gram-positive organisms and Enterobacteriaceae Selective for Salmonella and Shigella spp. except S. typhi.
Thayer-Martin agar
(modified Thayer Martin)
Blood agar base enriched with hemoglobin and supplement B; contaminating organisms inhibited by colistin, nystatin, vancomycin, and trimethoprim Selective for N. gonorrhoeae and N. meningitidis.
Supports the growth of Francisella and Brucella spp.
Thioglycollate broth Pancreatic digest of casein, soy broth, and glucose enrich growth of most microorganisms; includes reducing agents thioglycolate, cystine, and sodium sulfite; semisolid medium with a low concentration of agar reducing oxygen diffusion in the medium Supports growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms
Thiosulfate citrate-bile salts (TCBS) agar Peptone base agar with yeast extract, bile salts, citrate, sucrose, ferric citrate, and sodium thiosulfate; bromthymol blue acts as indicator Selective and differential for Vibrio spp.
Todd-Hewitt broth supplemented with antibiotics (LIM) Todd-Hewitt, an enrichment broth for streptococci, is supplemented with nalidixic acid and gentamicin or colistin for greater selectivity; thioglycollate and agar reduce redox potential Selection and enrichment for Streptococcus agalactiae in female genital specimens
Trypticase soy broth (TSB) All-purpose enrichment broth that can support the growth of many fastidious and nonfastidious bacteria Enrichment broth used for subculturing various bacteria from primary agar plates
Xylose lysine desoxycholate (XLD) agar Yeast extract agar with lysine, xylose, lactose, sucrose, and ferric ammonium citrate; sodium desoxycholate inhibits gram-positive organisms; phenol red as indicator Isolation and differentiation of Salmonella and Shigella spp. from other gram-negative enteric bacilli

Of the dozens of available media, only those most commonly used for routine diagnostic bacteriology are summarized in this discussion. Part VII discusses which media should be used to culture bacteria from various clinical specimens. Similarly, other chapters throughout Part III discuss media used to identify and characterize specific organisms.

Chocolate Agar.

Chocolate agar is essentially the same as blood agar except that during preparation the red blood cells are lysed when added to molten agar base. The cell lysis provides for the release of intracellular nutrients such as hemoglobin, hemin (“X” factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or “V” factor) into the agar for utilization by fastidious bacteria. Red blood cell lysis gives the medium a chocolate-brown color from which the agar gets its name. The most common bacterial pathogens that require this enriched medium for growth include Neisseria gonorrhoeae, the causative agent of gonorrhea, and Haemophilus spp., which cause infections usually involving the respiratory tract and middle ear. Neither of these species is able to grow on sheep blood agar.

Gram-Negative (GN) Broth.

A selective broth, gram-negative (GN) broth is used for the cultivation of gastrointestinal pathogens (i.e., Salmonella spp. and Shigella spp.) from stool specimens and rectal swabs. The broth contains several active ingredients, including sodium citrate and sodium desoxycholate (a bile salt), that inhibit gram-positive organisms and the early multiplication of gram-negative, nonenteric pathogens. The broth also contains mannitol as the primary carbon source. Mannitol is the favored energy source for many enteric pathogens, but it is not utilized by many other nonpathogenic enteric organisms. To optimize its selective nature, GN broth should be subcultured 6 to 8 hours after initial inoculation and incubation. After this time, the nonenteric pathogens begin to overgrow the pathogens that may be present in very low numbers.

Hektoen Enteric (HE) Agar.

Hektoen enteric (HE) agar contains bile salts and dyes (bromthymol blue and acid fuchsin) to selectively slow the growth of most nonpathogenic gram-negative bacilli found in the gastrointestinal tract and allow Salmonella spp. and Shigella spp. to grow. The medium is also differential because many non-enteric pathogens that do grow will appear as orange to salmon-colored colonies. This colony appearance results from the organism’s ability to ferment the lactose in the medium, resulting in the production of acid, which lowers the medium’s pH and causes a change in the pH indicator bromthymol blue. Salmonella spp. and Shigella spp. do not ferment lactose, so no color change occurs and their colonies maintain the original blue-green color of the medium. As an additional differential characteristic, the medium contains ferric ammonium citrate, an indicator for the detection of H2S, so that H2S-producing organisms, such as Salmonella spp., can be visualized as colonies exhibiting a black precipitate (Figure 7-6).

Sheep Blood Agar.

Most bacteriology specimens are inoculated to sheep blood agar plates because this medium supports growth for all but the most fastidious clinically significant bacteria. Additionally, the colony morphologies that commonly encountered bacteria exhibit on this medium are familiar to most clinical microbiologists. The medium consists of a base containing a protein source (e.g., tryptones), soybean protein digest (containing a slight amount of natural carbohydrate), sodium chloride, agar, and 5% sheep blood.

Certain bacteria produce extracellular enzymes that lyse red blood cells in the agar (hemolysis). This activity can result in complete clearing of the red blood cells around the bacterial colony (beta hemolysis) or in only partial lysis of the cells to produce a greenish discoloration around the colony (alpha hemolysis). Other bacteria have no effect on the red blood cells, and no halo is produced around the colony (gamma or nonhemolytic). Microbiologists often use colony morphology and the degree or absence of hemolysis as criteria for determining what additional steps will be necessary for identification of a bacterial isolate. To read the hemolytic reaction on a blood agar plate accurately, the technologist must hold the plate up to the light and observe the plate with the light coming from behind (i.e., transmitted light).

Modified Thayer-Martin Agar.

Modified Thayer-Martin (MTM) agar is an enrichment and selective medium for the isolation of Neisseria gonorrhoeae, the causative agent of gonorrhea, and Neisseria meningitidis, a life-threatening cause of meningitis from specimens containing mixed flora. The enrichment portion of the medium is the basal components and the chocolatized blood, while the addition of antibiotics provides a selective capacity. The antibiotics include colistin to inhibit other gram-negative bacteria, vancomycin to inhibit gram-positive bacteria, and nystatin to inhibit yeast. The antimicrobial trimethoprim is also added to inhibit Proteus spp., which tend to swarm over the agar surface and mask the detection of individual colonies of the two pathogenic Neisseria spp. A further modification, Martin-Lewis agar, substitutes ansamycin for nystatin and has a higher concentration of vancomycin.

Thioglycollate Broth.

Thioglycollate broth is the enrichment broth most frequently used in diagnostic bacteriology. The broth contains many nutrient factors, including casein, yeast and beef extracts, and vitamins, to enhance the growth of most medically important bacteria. Other nutrient supplements, an oxidation-reduction indicator (resazurin), dextrose, vitamin K1, and hemin have been used to modify the basic thioglycollate formula. In addition, this medium contains 0.075% agar to prevent convection currents from carrying atmospheric oxygen throughout the broth. This agar supplement and the presence of thioglycolic acid, which acts as a reducing agent to create an anaerobic environment deeper in the tube, allow anaerobic bacteria to grow.

Gram-negative, facultatively anaerobic bacilli (i.e., those that can grow in the presence or absence of oxygen) generally produce diffuse, even growth throughout the broth, whereas gram-positive cocci demonstrate flocculation or clumps. Strict aerobic bacteria (i.e., require oxygen for growth), such as Pseudomonas spp., tend to grow toward the surface of the broth, whereas strict anaerobic bacteria (i.e., those that cannot grow in the presence of oxygen) grow at the bottom of the broth (Figure 7-7).

Xylose-Lysine-Desoxycholate (XLD) Agar.

As with HE agar, xylose-lysine-desoxycholate (XLD) agar is selective and differential for Shigella spp. and Salmonella spp. The salt, sodium desoxycholate, inhibits many gram-negative bacilli that are not enteric pathogens and inhibits gram-positive organisms. A phenol red indicator in the medium detects increased acidity from carbohydrate (i.e., lactose, xylose, and sucrose) fermentation. Enteric pathogens, such as Shigella spp., do not ferment these carbohydrates, so their colonies remain colorless (i.e., the same approximate pink to red color of the un-inoculated medium). Even though they often ferment xylose, colonies of Salmonella spp. are also colorless on XLD, because of the decarboxylation of lysine, which results in a pH increase that causes the pH indicator to turn red. These colonies often exhibit a black center that results from Salmonella spp. producing H2S. Several of the nonpathogenic organisms ferment one or more of the sugars and produce yellow colonies (Figure 7-8).

Preparation of Artificial Media

Nearly all media are commercially available as ready-to-use agar plates or tubes of broth. If media are not purchased, laboratory personnel can prepare agars and broths using dehydrated powders that are reconstituted in water (distilled or deionized) according to manufacturer’s recommendations. Generally, media are reconstituted by dissolving a specified amount of media powder, which usually contains all necessary components, in water. Boiling is often required to dissolve the powder, but specific manufacturer’s instructions printed in media package inserts should be followed exactly. Most media require sterilization so that only bacteria from patient specimens will grow and not contaminants from water or the powdered media. Broth media are distributed to individual tubes before sterilization. Agar media are usually sterilized in large flasks or bottles capped with either plastic screw caps or plugs before being placed in an autoclave.

Media Sterilization.

The timing of autoclave sterilization should start from the moment the temperature reaches 121° C and usually requires a minimum of 15 minutes. Once the sterilization cycle is completed, molten agar is allowed to cool to approximately 50° C before being distributed to individual petri plates (approximately 20 to 25 mL of molten agar per plate). If other ingredients are to be added (e.g., supplements such as sheep blood or specific vitamins, nutrients, or antibiotics), they should be incorporated when the molten agar has cooled, just before distribution to plates.

Delicate media components that cannot withstand steam sterilization by autoclaving (e.g., serum, certain carbohydrate solutions, certain antibiotics, and other heat-labile substances) can be sterilized by membrane filtration. Passage of solutions through membrane filters with pores ranging in size from 0.2 to 0.45 µm in diameter will not remove viruses but does effectively remove most bacterial and fungal contaminants. Finally, all media, whether purchased or prepared, must be subjected to stringent quality control before being used in the diagnostic setting (for more information regarding quality control see Chapter 79).

Cell Cultures.
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